Fenton-promoted luminol ECL imaging method for multiple enzyme activities

Abstract

The invention relates to a Fenton-promoted luminol electrochemical luminescence imaging method, which is used for realizing spatial mapping of various enzyme activities in a single tissue slice. The method quantitatively visualizes the activity of a single enzyme by combining enzymatic conversion of a substrate and chemical limitation of locally produced hydrogen peroxide. In order to limit diffusion of hydrogen peroxide for high-resolution spatial imaging, iron oxide nanoparticles are coated on the surface of a tissue to start a Fenton process, and hydrogen peroxide is locally converted into hydroxyl radicals with short service life within a limited diffusion distance.

Description

Technical Field

[0001] The present application relates to the field of electrochemical analysis, and in particular to a Fenton-promoted luminol ECL imaging method for multiple enzyme activities. Background Art

[0002] The spatial characteristics of molecular parameters in intact tissue samples provide important information along with their spatial location, which helps to determine their functions in diseases. This emerging spatial research is expected to promote a new generation of scientific discoveries. Current research and related technologies focus on the spatial analysis of protein expression, gene mutations, chromatin structure and genome organization. It is well known that enzymes, as efficient protein machines, can accelerate substrate conversion at the molecular level and induce downstream biological effects. The spatial analysis of enzyme activity in tissue sections is crucial for understanding the complex metabolic and signaling networks under disease conditions. However, existing spatial research technologies, including barcoding with reporters, immunohistochemistry and fluorescence in situ hybridization, are mainly used for protein abundance analysis, making them unsuitable for quantitative enzyme activity determination. To address this challenge, activity-based protein analysis has been developed to visualize enzyme activities in cells or tissues with high-throughput information. However, the development of these probes is usually expensive and probe labeling is time-consuming. In addition, the widespread use of fluorescent tags introduces problems such as background interference, tissue autofluorescence, phototoxicity and photobleaching, which affect quantitative and sensitive measurements. Therefore, the development of a robust method to sensitively spatially visualize multiple enzyme activities in tissue sections remains a pressing need.

[0003] Electrochemiluminescence (ECL) refers to the process in which a luminophore undergoes an electron transfer reaction on the electrode surface to generate an excited state, which then relaxes to the ground state and emits light. Given its unique advantages, such as zero luminescence background and controllable spatiotemporal properties, ECL-based detection offers high sensitivity and high throughput in bioanalysis and clinical testing. Various ECL visualization protocols have been developed for biological samples, including single cells, organelles, liposomes, and single protein molecules, to provide spatial information with a high signal-to-noise ratio. Luminol / hydrogen peroxide (H 2 O 2 ) combination is a water-soluble ECL system operating at low potential, in which H 2 O 2 As a co-reactant, it promotes the ECL emission of luminol. Prior art has reported the use of enhanced luminol ECL to electrochemically visualize H at electrodes. 2 O 2 In addition, oxidases are introduced to oxidize the corresponding substrates (such as glucose and cholesterol) to produce H 2 O 2 Since the substrate concentration directly affects the amount of H produced 2 O 2The amount of H and the resulting ECL emission intensity, the recorded ECL images can quantitatively display the distribution of targeted biomolecules in single living cells. 2 O 2 , this strategy should enable visualization of enzyme activity in such complex samples. However, unlike single cells, which are simple and micrometer-sized, tissue sections are millimeter-scale in size, only a few micrometers thick, and have complex structures. H generated at the tissue / solution interface 2 O 2 It can diffuse rapidly into the tissue and is not consumed in situ in the tissue where it is produced, but is oxidized on the electrode surface to form non-spatial specific signals. This phenomenon affects the accuracy of enzyme activity signal localization and makes tissue imaging challenging. Summary of the invention

[0004] In order to solve the problem of accuracy of enzyme activity signal detection and positioning in the prior art, the present application provides a Fenton-promoted luminol ECL imaging method for multiple enzyme activities, comprising the following steps:

[0005] Step 1, tissue slice preparation, the tissue slice to be tested is warmed to room temperature, washed with PBS to remove OCT, and transferred to the ITO electrode surface;

[0006] Step 2, preparation of Fe 3 O 4 Nafion coating, Fe 3 O 4 Nafion dispersion was coated on the tissue section on the ITO electrode and dried in an oven to form a uniform Fe 3 O 4 Nafion coating;

[0007] Step 3, tissue section enzyme activity imaging, start the first cycle, in the first cycle, add L012 to the reaction chamber, apply a potential switch on the ITO electrode to capture ECL base1 Image, using EMCCD to record ECL background as ECL base images, as a baseline;

[0008] Step 4, reapply the potential switch to capture ECL base2 Images were obtained from ECL using Image J. base2 Subtract ECL base1 And divide the result by ECL base1 To process the image to obtain ΔECL baseImage, the image is qualified without light signal, add the target enzyme specific substrate to be detected into the reaction chamber, wait for 1 minute, and then apply the potential switch again to capture the ECL signal image ECL signal , images were processed using Image J, and ECL signal Image subtracted ECL base2 image, and divide the result by ECL base2 images to obtain enzyme-specific ECL enhancement images, with the ΔECL value corresponding to the target enzyme activity on the tissue sections, the reaction solution was removed to stop the LOX catalytic reaction, and then the reaction chamber was washed with 100 μL PBS to remove the residual reaction solution;

[0009] Step 5, sequential imaging of multiple enzymes on the tissue section, starting the second cycle by adding fresh L012, repeating steps 3 and 4 for the second cycle, adding multiple target enzymes in sequence, and repeating the cycle to obtain ΔECL images of multiple enzymes in the same tissue section area.

[0010] The present application also provides an application of the detection method in imaging detection of enzyme activity in tissues.

[0011] Beneficial Effects

[0012] Individual enzyme activities were quantitatively visualized by combining enzymatic conversion of substrates with chemical confinement of locally generated hydrogen peroxide. To restrict the diffusion of hydrogen peroxide (centimeter scale) for high-resolution spatial imaging, FeO3 nanoparticles were coated on the tissue surface to initiate the Fenton process, which locally converts long-lived hydrogen peroxide into short-lived hydroxyl radicals within a limited diffusion distance (55-175nm). Fenton-facilitated ECL emission was confined to the site of enzyme conversion, providing unprecedented spatial visualization of four tumor-associated oxidases within a single tissue section. Spatial colocalization of the oxidases revealed synergistic effects of lysyl oxidase and aescin sulfhydryl oxidase on post-translational modification of tumor extracellular matrix proteins, as well as a previously undiscovered interaction with amiloride-sensitive amine oxidase that could not be distinguished based on expression or single enzyme activity alone. This approach provides a new activity-based protein analysis tool at the tissue level, providing new data for future enzymomic studies and multimodal imaging.

[0013] The activities of multiple oxidases within a single tissue section can be visualized by Fenton-facilitated ECL microscopy. Hydrogen peroxide produced by the enzyme reaction enhances the ECL intensity of L012, enabling ΔECL to reveal enzyme activity and its spatial distribution. By adopting the Fenton process to convert long-lived hydrogen peroxide into short-lived and highly reactive hydroxyl radicals, the diffusion of the ΔECL signal is confined to the nanoscale, achieving a high degree of spatial resolution. Through continuous imaging of different oxidase activities and co-localization analysis of expression and activity, abnormal activation of tumor-related proteins that cannot be achieved by traditional immunoassays was discovered. This method provides new insights into the diffusion problem of ECL signals and helps to further improve the spatial resolution of ECL imaging. More importantly, this protocol may open up a direction for comprehensive studies of multiple enzyme activities at the tissue level and provide new data for future enzymomic studies and multimodal imaging. Ultimately, a comprehensive understanding of enzymes in living systems will provide a new approach to identify therapeutic targets and develop small molecule inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram showing the principle of detection of this application

[0015] Figure 2 The principle diagram of the detection of this application is shown

[0016] Figure 3 Schematic diagram and empirical diagram showing the principle of Fenton promotion in this application

[0017] Figure 4 Flow chart and demonstration diagram showing various enzyme activity imaging of this application

[0018] Figure 5 Shows an application example of the detection of this application DETAILED DESCRIPTION

[0019] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] An embodiment of the present application provides a Fenton-promoted luminol ECL imaging method for multiple enzyme activities, characterized in that it includes the following steps:

[0023] Step 1, tissue slice preparation, the tissue slice to be tested is warmed to room temperature, washed with PBS to remove OCT, and transferred to the ITO electrode surface;

[0024] Step 2, preparation of Fe 3 O 4 Nafion coating, Fe 3 O 4 Nafion dispersion was coated on the tissue section on the ITO electrode and dried in an oven to form a uniform Fe 3 O 4 Nafion coating;

[0025] Step 3, tissue section enzyme activity imaging, start the first cycle, in the first cycle, add L012 to the reaction chamber, apply a potential switch on the ITO electrode to capture ECL base1 Image, using EMCCD to record ECL background as ECL base images, as a baseline;

[0026] Step 4, reapply the potential switch to capture ECL base2 Images were obtained from ECL using Image J. base2 Subtract ECL base1 And divide the result by ECL base1 To process the image to obtain ΔECL base Image, the image is qualified without light signal, add the target enzyme specific substrate to be detected into the reaction chamber, wait for 1 minute, and then apply the potential switch again to capture the ECL signal image ECL signal , images were processed using Image J, and ECL signal Image subtracted ECL base2 image, and divide the result by ECL base2 images to obtain enzyme-specific ECL enhancement images, with the ΔECL value corresponding to the target enzyme activity on the tissue sections, the reaction solution was removed to stop the LOX catalytic reaction, and then the reaction chamber was washed with 100 μL PBS to remove the residual reaction solution;

[0027] Step 5, sequential imaging of multiple enzymes on the tissue section, starting the second cycle by adding fresh L012, repeating steps 3 and 4 for the second cycle, adding multiple target enzymes in sequence, and repeating the cycle to obtain ΔECL images of multiple enzymes in the same tissue section area.

[0028] In one embodiment, in the imaging method, an ITO electrode is used as the working electrode, a platinum wire is used as the counter electrode, an Ag / AgCl electrode is used as the reference electrode, a silicone O-ring is fixed to the conductive surface of the ITO electrode by polydimethylsiloxane and used as a reaction chamber, a potential switch is applied using a CHI 600E electrochemical analyzer, ECL imaging and bright field imaging are performed using an epifluorescence microscope equipped with an electron multiplying charge coupled device (EMCCD) camera, an MPI-A type ECL analyzer is used to collect cyclic voltammetry and ECL signals, and a JSM-7800F instrument is used to obtain scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) images.

[0029] In one embodiment, in step 2, Fe 3 O 4 The Nafion dispersion was prepared by diluting the Nafion dispersion with anhydrous ethanol to produce a 0.5% w / w dispersion, washing the Fe 3 O 4 Nanoparticles, and Fe 3 O 4 The nanoparticles were resuspended in anhydrous ethanol to form a 1% w / v suspension. After sonication, the suspension was mixed with Nafion dispersion at a volume ratio of 1:1 and sonicated to obtain Fe 3 O 4 -Nafion dispersion.

[0030] In one embodiment, Fe 3 O 4 Nafion dispersion was coated on the tissue section on the ITO electrode to 3 O 4 A 10 μL sample of Nafion dispersion was evenly coated on the surface of the tissue section on the ITO electrode and dried in an oven at 37 °C for 1 min to form a uniform Fe 3 O 4 Nafion coating.

[0031] In one embodiment, tissue slice enzyme activity imaging, L012 is added to the reaction chamber, a potential switch is applied to the ITO electrode, and the ECL background is recorded using an EMCCD as ECL base image, as a baseline, then, enzyme-specific substrates were added to the reaction chamber, and the same potential switch was applied to capture the ECL signal for ECL signal Images, Image processing was performed using Image J, and ECL signal Image subtracted ECL base image, and divide the result by ECL baseImages were taken to obtain enzyme-specific ECL enhancement ΔECL images and obtain the ECL light emission change rate related to enzyme activity.

[0032] In one embodiment, in step 3, the tissue section enzyme activity imaging is performed by examining the tissue section under the bright field of a microscope and recording the image using a common CCD camera.

[0033] In one embodiment, in step 5, a specific substrate corresponding to the target enzyme is introduced to obtain a ΔECL image indicating the activity of the target enzyme. After completing the imaging of a single target enzyme activity, step 4 is implemented, the reaction is terminated and the reaction system is reset, and steps 3 and 4 are repeated to sequentially complete the imaging of multiple target enzyme activities on a single tissue section.

[0034] In one embodiment, in step 5, the plurality of enzymes include LOX, QSOX, NOX and DAO.

[0035] In one embodiment, step 5 includes four cycles, with a washing step between each cycle, and ΔECL base Images should be blank and recorded at the beginning of each cycle to ensure that the reaction system is reset for each cycle.

[0036] In one embodiment, in step 5, the substrates used in the four cycles are L-lysine, dithiothreitol, histamine and nicotinamide adenine dinucleotide phosphate, respectively.

[0037] An embodiment of the present application provides the application of the detection method in imaging detection of enzyme activity in tissues.

[0038] 1. Reagents

[0039] 1x phosphate buffer solution (PBS, pH 7.2-7.4) was from Solarbio. 10% formalin was from Citotest. Cryosection embedding medium (OCT) was from Sakura. Anhydrous ethanol and sucrose were provided by Sinopharm Chemical Reagent Co., Ltd. 8-Amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione (L012) was obtained from Wako PureChemical Co., Ltd and prepared into a 200 μM PBS solution. Perfluorosulfonic acid-PTFE copolymer (Nafion dispersion, 5% w / w in 1-propanol solution) was obtained from Alfa Aesar. Fe 3 O 4Nanoparticles (10 nm in diameter, 10 mg / mL) were from BaseLine ChromTech Research Center. Lysine oxidase (LOX) standard was purchased from Everest Biotech. 2.5% glutaraldehyde was supplied by Wabcan. Anti-LOX antibody was from Abcam, while QSOX, NOX and DAO antibodies were from Proteintech. L-lysine, histamine, dithiothreitol and nicotinamide adenine dinucleotide phosphate were purchased from Aladdin, and these substrates were prepared as 100 mM stock solutions in PBS for experiments. β-Aminopropionitrile (BAPN) and dimethyl sulfoxide were from Sigma-Aldrich. The fluorescent dye 2',7'-dichlorodihydrofluorescein (DCFH) for reactive oxygen species (ROS) detection was from Heowns.

[0040] 2. Instrumentation

[0041] The working electrode used was indium tin oxide coated glass (ITO electrode, sheet resistance <10 ohm / sq) produced by Zhuhai Kaiwei Optoelectronics Technology Co., Ltd., a platinum wire was used as the counter electrode, and an Ag / AgCl (3M KCl) electrode was used as the reference electrode. A silicone O-ring (inner diameter 8 mm) was inserted through a polydimethylsiloxane (Dow Corning ) is fixed on the conductive surface of the ITO electrode and used as a reaction chamber. Potential is applied using a CHI 600E electrochemical analyzer (Shanghai Chenhua Instrument Co., Ltd.). ECL imaging and bright field imaging are performed using an epifluorescence microscope (Eclipse Ti2, Nikon) equipped with an electron multiplying charge coupled device camera (Evolve 512Delta, Photometrics). Observation is performed using a 4x objective lens (NA0.13). Cyclic voltammetry and ECL signals are collected using an MPI-A type ECL analyzer (Xi'an Ruimai Analytical Instrument Co., Ltd.). Scanning electron microscopy (SEM) and energy dispersive x-ray spectroscopy (EDS) images are obtained using a JSM-7800F instrument (JEOL, Ltd). Nanoparticle tracking analysis (NTA) is performed by NanoSight Pro (Malvern Panalytical). Frozen tissue blocks are sliced ​​using a cryostat (CM1950, Leica).

[0042] 3. Tissue Collection

[0043] Volunteer patients with pancreatic space-occupying lesions who met surgical indications were recruited from the Second Affiliated Hospital of Zhejiang University School of Medicine. The Human Research Ethics Committee of the Second Affiliated Hospital of Zhejiang University approved the study (certificate number: 2019-447). This study was conducted in accordance with relevant regulations and guidelines, including the ethical principles for medical research involving human subjects designed by the World Medical Association Declaration of Helsinki. Tissue blocks from patients were obtained during surgery and preserved in 10% cold formalin.

[0044] 4. Preparation of Tissue Sections

[0045] The fixed tissue blocks were dehydrated in a gradient of 15% and 30% sucrose solutions before embedding in OCT complex. They were then cut into 6-μm sections using a cryostat at -22°C. The tissue sections were warmed to room temperature, washed with PBS to remove OCT, and carefully transferred to the ITO electrode surface for subsequent use.

[0046] 5. Organizational Fe 3 O 4 Preparation of Nafion coating

[0047] The Nafion dispersion was diluted with absolute ethanol to produce a 0.5% w / w dispersion. The Fe 3 O 4 The nanoparticles were resuspended in anhydrous ethanol to form a 1% w / v suspension. After ultrasonic treatment for 10 minutes, the suspension was mixed with Nafion dispersion at a volume ratio of 1:1. The mixture was then subjected to an ultrasonic bath for 30 minutes to obtain Fe 3 O 4 -Nafion dispersion. The particle concentration was determined to be 4.67×10 8 A 10 μL aliquot of this dispersion was evenly applied to the tissue section on the ITO electrode and dried in an oven at 37 °C for 1 min to form a uniform Fe 3 O 4 Nafion coating.

[0048] 6. Enzyme Activity Imaging in Tissue Sections

[0049] The process begins by examining the tissue section under bright field on a microscope and recording the image using a regular CCD camera. Then, 80 μL of 200 μM L012 is added to the reaction chamber, ensuring that all three electrodes are in contact with the solution. A switching potential of 1.0 to -1.0 V (vs. Ag / AgCl) is applied to the ITO electrode and the ECL is recorded using an EMCCD. baseImage (as a baseline). Then, add 4 μL of 100 mM enzyme-specific substrate (e.g., L-lysine for LOX) to the reaction chamber. Wait 1 minute and then apply the same switching potential to capture the ECL signal image. Image processing using Image J: Subtract the ECL from the ECL signal image base image, and then divide the result by the ECL base , and the ECL light emission change rate (ΔECL image) related to enzyme activity was obtained. The exposure time of all ECL images was 5 seconds.

[0050] 7. Imaging of LOX Standard Enzyme Activity

[0051] To prepare the LOX standard, a 0.5 ng / mL LOX solution was prepared using 2.5% glutaraldehyde. 0.5 μL of this solution was dropped onto the ITO electrode and dried at 37°C to obtain an electrode with a solid LOX standard. The imaging process then followed the same procedure outlined in point 6.

[0052] 8. Active oxygen detection

[0053] Tissue sections were immersed in PBS with 200 μM DCFH probe. FITC filter (λ exc :465-495,DM:505,λ em :512-558) record FL base Image (signal comes from tissue autofluorescence). Then, 4 μL of 100 mM L-lysine was added to trigger the LOX catalytic reaction. After waiting for 1 minute, the FL was recorded using the same filter. signal Image J was used to obtain the images (i.e. tissue autofluorescence plus the fluorescence response of the DCFH probe to enzyme-derived ROS). signal Subtract FL from the image base The images were processed to obtain the FL light emission change rate corresponding to the ROS content (ΔFL image).

[0054] 9. Imaging of multiple enzyme activities on tissue sections

[0055] This section outlines the sequential imaging of four enzymes (LOX, QSOX, NOX, and DAO) on tissue sections. In the first cycle, 80 μL of 200 μM L012 was added to the reaction chamber. A switching potential of 1.0 to -1.0 V (vs. Ag / AgCl) was applied to the ITO electrode to capture ECL base1 After 1 minute, the potential was reapplied to capture the ECL base2 Image. Image J was used to obtain base2 Subtract ECL base1And divide the result by ECL base1 To process the image to obtain ΔECL base Image, in the absence of light signal, the image is considered qualified. Next, 4 μL of 100 mM L-lysine was added to the reaction chamber. After waiting for 1 minute, the potential switch was applied again to capture the ECL signal image. These images were processed with Image J: signal Subtract ECL from the image base2 Image, divided by ECL base2 , to calculate the ECL light emission change rate (ΔECL oxidase Image), which corresponds to the LOX activity on the tissue. The reaction solution was removed to stop the LOX catalytic reaction, and then the reaction chamber was washed with 100 μL PBS for 1 minute to remove the residual reaction solution. The second cycle was started by adding fresh 80 μL 200 μM L012. This process was repeated for all four cycles to obtain the ΔECL of LOX, QSOX, NOX, and DAO in the same tissue section area. oxidase Each cycle is only in ΔECL base The blank was performed to ensure the successful initialization of the reaction system. The substrates used in the four cycles were L-lysine, dithiothreitol, histamine, and nicotinamide adenine dinucleotide phosphate. The exposure time of all ECL images was set to 5 seconds. The colocalization of the ΔECL oxidase signals of the four enzymes was analyzed using the JACoP plugin in Image J.

[0056] 10. Enzyme Expression Imaging

[0057] The multiple labeling immunofluorescence service for tissue sections is provided by Ruchuang Biotechnology Co., Ltd. The samples are adjacent tissues for multiple enzyme activity ECL imaging. Since frozen sections are difficult to maintain morphology during multiple immunofluorescence staining processes, the tissues are prepared as paraffin sections. The paraffin sections are dewaxed by washing in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 85% ethanol, 75% ethanol and distilled water for 15 minutes in sequence. Antigen retrieval is performed by placing the sections in EDTA antigen retrieval buffer and heating them in a microwave oven. After natural cooling, the sections are washed three times with PBS (pH 7.4). They are then incubated in 3% hydrogen peroxide (H2O2) at room temperature in the dark. 2 O 2) solution for 25 minutes to block endogenous peroxidase. After washing three times with PBS, the sections were incubated with 3% BSA-PBS at room temperature for 30 minutes. After removing the blocking solution, anti-LOX antibody diluted with antibody diluent was added, and the sections were incubated at 37°C for 1 hour. The sections were then washed three times with PBS, with each wash lasting 5 minutes. Then, the tissue was covered with HRP secondary antibody, incubated in the dark at room temperature for 50 minutes, and washed three times with PBS. The tissue was stained with TYR620 fluorescent dye for 15 minutes and washed 3 times with PBS. These steps completed the immunofluorescence staining of LOX. The steps from antigen retrieval to fluorescent dye application were repeated to label the other three enzymes. Different primary antibodies and fluorescent dyes were used for each enzyme to distinguish them: QSOX corresponds to TYR570, NOX corresponds to TYR520, and DAO corresponds to TYR690. The sections were then counterstained with DAPI dye solution for 10 minutes to highlight the nuclei, and then washed three times with PBS. The sections were slightly dried and mounted with anti-fluorescence quenching tablets. Finally, the sections were observed and imaged using a multi-channel fluorescence scanner. The colocalization of the fluorescence signals of the four enzymes was analyzed using the JACoP plugin in Image J.

[0058] 11. Hematoxylin and eosin (H&E) staining

[0059] H&E staining service for tissue sections was provided by Ruchuang Biotechnology Co., Ltd. The staining procedure included the following steps: hematoxylin staining for 5 minutes, differentiation in 1% hydrochloric acid for 5 seconds, blueing with 1% ammonia for 10 seconds, and eosin staining for 1 minute. The stained sections were then dehydrated in a gradient through different concentrations of ethanol (90% ethanol, 100% ethanol I, 100% ethanol II), followed by treatment with xylene I and xylene II. After sealing with neutral resin, the tissue was imaged using a white light scanner.

[0060] 12. Data Analysis and Visualization

[0061] The intensity values ​​of ECL and fluorescence were extracted by Image J.

[0062] Experimental Principle: Imaging of LOX activity in tissue sections without the aid of Fenton reaction. Lysyl oxidase (LOX), which is widely present in human tissues and is considered a therapeutic target, was selected as a model enzyme. Indium tin oxide (ITO) electrode was selected as the working electrode due to its optical transparency and high performance in luminol ECL. In this application, the luminol analog L012 (8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione) was used due to its high ECL efficiency at physiological pH. L012 was oxidized and electrode passivation was reduced by applying a switching potential switch between 1.0 and -1.0 V (the potential is relative to the Ag / AgCl reference electrode). Prior to imaging the tissue, LOX standards (purified form of the enzyme) were first immobilized on the ITO surface using glutaraldehyde to simulate the enzyme state in tissue sections. The high affinity LOX substrate L-lysine was selected to initiate the catalytic reaction. LOX catalyzes the reaction to produce H 2 O 2 , and finally produce ECL.

[0063] L-lysine + H 2 O + O 2 → 6-amino-2-oxohexanoate + H 2 O 2 (1)

[0064]

[0065] HO 2 - – e - → HO 2 · (3)

[0066]

[0067] LH - – e - → LH · (6)

[0068]

[0069] L ·- + O 2 ·- → LO 2 2- → 3AP * + N 2 (8)

[0070] 3AP *→ 3AP + hv (9)

[0071] Where L is L012 and 3AP is 3-aminophthalate.

[0072] To isolate the enzyme-specific ECL signal, base ) and after (ECL signal ) records the ECL emission. signal Subtract ECL base And divide it by ECL base , the ECL light emission change rate (ΔECL) is used to indicate enzyme activity ( Figure 2 The ΔECL positive signals of LOX spots (containing 0.5 ng / mL LOX) on the ITO surface are consistent ( Figure 2 (B) The ECL intensity increases with increasing enzyme and substrate concentrations, which is a typical feature of enzyme reactions. These results confirm that the ΔECL value of the enzyme spot area is correlated with the activity of LOX in catalyzing the oxidative deamination of L-lysine. This suggests that the same strategy can be applied to imaging enzyme activity in more complex samples such as biological tissue sections.

[0073] To image tissue sections, freshly prepared frozen sections were used in this application example to maintain maximum enzyme activity. The section thickness was 6 μm, allowing the diffusion range of the L012 intermediate oxidized and deprotonated under neutral conditions to be within the range of the diffusion of the L012 intermediate and H 2 O 2 The coexisting reaction layers have sufficient spatial overlap. Endogenous LOX in tissues reacts with substrates to produce H 2 O 2 , H 2 O 2 As a physiologically derived co-reactant, it locally enhances the light emission of ECL. Prior to ECL imaging, fluorescence experiments were performed to confirm the presence of H on tissues after the addition of L-lysine. 2 O 2 After ECL imaging, by comparing with the bright field image, it was confirmed that the ΔECL signal was concentrated in the tissue area ( Figure 2 C). Blocking LOX activity by adding the LOX inhibitor β-aminopropionitrile resulted in the disappearance of the ΔECL signal on the tissue. Thus, a direct correlation between ΔECL and LOX activity was confirmed. However, a scattered ΔECL signal was observed in the bare electrode area surrounding the tissue ( Figure 2 (D) These signals extend over 100 μm and are attributed to H 2 O 2 Diffusion across the electrodes. This diffusion causes crosstalk and obscures the precise localization of the active enzyme.

[0074] The ΔECL signal essentially reflects the H 2 O 2 The distribution of H 2 O 2 The diffusion of L012 and H determines the spatial resolution of ECL microscopy. 2 O 2 It is electro-oxidized at 1V to form L012 anion radical (L ·- ) and superoxide anion (O 2 ·- )( Figure 1 , Route I). It is known that O 2 ·- The half-life is ~0.1 ms. Therefore, the lifetime can be converted into a diffusion distance, which is calculated to be about 0.5 μm (the diffusion coefficient is 1.0×10 -5 cm 2 / s). This means that the signal diffusion of ΔECL at the bottom of the tissue slice is negligible in large field imaging. Therefore, the ECL process at the bottom of the tissue slice does not reduce the spatial resolution. However, a large amount of H 2 O 2 , with a half-life of more than 2 days. Since the upper side of the tissue is exposed to the bulk solution, H 2 O 2 It cannot immediately contact the electrode surface and be consumed. Instead, it diffuses in the solution over a distance of centimeters (with a diffusion coefficient of 1.8×10 -5 cm 2 / s) until it is oxidized on the bare electrode surface. On the electrode surface, H 2 O 2 With L ·- The reaction produces ΔECL outside the enzyme conversion site ( Figure 2 (E) This non-in-situ signal reduces the resolution of enzyme activity imaging in tissues. Therefore, the present application implements coating a Fenton reaction layer on the surface of tissue slices to eliminate non-in-situ signals and improve imaging resolution.

[0075] High-resolution imaging of LOX activity by optimizing the Fenton process. In order to achieve high spatial resolution ECL imaging of enzyme activity, Fe 3 O 4 Nanoparticles are coated on the tissue surface to initiate the Fenton process. The Fe 3 O 4 / H 2 O 2 The system has been used in a variety of fields including chemical analysis, biosensing, molecular synthesis, and environmental remediation. 2+ / Fe 3+ In the presence of H 2 O2 The reaction mechanism of the decomposition is shown in equations (10) and (11).

[0076] Fe 2+ +H 2 O 2 →Fe 3++· OH+OH - k=40-80M -1 s -1 (10)

[0077] Fe 3+ +H 2 O 2 →Fe 2+ +HO 2 · +H + k=0.001-0.01M -1 s -1 (11)

[0078] In this process, H 2 O 2 Acts as an oxidant, while iron ions act as catalysts, generating highly reactive ROS. 2+ / Fe 3+ and H 2 O 2 The reaction rate between them is , and the products of the Fenton process are mainly hydroxyl radicals ( · OH). · OH is a short-lived ROS with a half-life of 13 μs and a diffusion distance of ~200 nm in aqueous solution (D = 2.3 × 10 -5 cm 2 / s). Therefore, the long-lived H 2 O 2 Transformed into short-lived and highly active · OH can confine the ECL light emission to the reaction site catalyzed by the enzyme.

[0079] In the present embodiment, the upper side of the tissue section is coated with Fe 3 O 4 Heterogeneous Fenton layer (2 μm thick) composed of nanoparticles and Nafion matrix. The nanoscale porous structure of Nafion membrane facilitates the contact between substrate and tissue and promotes H 2 O 2 and Fe 3 O 4 The interaction between nanoparticles. Fenton layer, tissue and electrodes together form a "sandwich" structure ( Figure 3 For this purpose, Fe with a diameter of 10 nm 3 O4 The nanoparticles were dispersed in 0.25% Nafion. In this way, the Fenton reaction can be accelerated in a confined space, improving the ECL efficiency and limiting the diffusion of ECL co-reactants. As observed, the coating maintained good transparency, allowing for clear observation of tissue sections under bright field microscopy ( Figure 3 (B) By adopting the "sandwich" structure, the diffuse ΔECL signal in the non-tissue area is eliminated, ensuring that the collected ΔECL signal corresponds to the precise reaction site of the enzyme in the tissue section. Figure 3 This strategy clearly captures the regional specificity of the distribution of enzyme activity in tissues, as shown in Figure C. Thus, Fenton coating greatly improves the spatial resolution of ECL microscopy and enables high-quality mapping of enzyme activity in tissues.

[0080] During imaging, two reaction pathways acting on the upper and lower surfaces of the tissue lead to the generation of ECL ( Figure 1 ). In both pathways, the luminophore originates from the free radicals generated by oxidation of L012 on the electrode surface. However, in route I, the co-reactant is H 2 O 2 O is generated by electrochemical reaction at the electrode 2 ·- , while in route II, the coreactant is H 2 O 2 Produced in the coating by Fenton reaction · OH. This means that the enzyme-derived H 2 O 2 are converted into short-lived ROS by electrochemical and Fenton processes, respectively. Essentially, the two “layers” in the “sandwich” intercept H 2 O 2 Diffusion from tissue sections into solution. It should be noted that these two pathways may not convert all enzymatic H produced by the tissue. 2 O 2 , especially H produced in tissue sections 2 O 2 However, since the tissue sections are only a few micrometers thick, the unconverted H 2 O 2 Lateral diffusion within the tissue is limited and unlikely to cause significant signal dispersion. Therefore, the captured ΔECL image can be viewed as a superposition of enzyme activity signals from the upper and lower surfaces of the tissue. This signal superposition along the micrometer-scale axis is common and acceptable in tissue section analysis methods such as H&E staining or immunohistochemistry.

[0081] Imaging of multiple enzyme activities on tissue sections. Tissues are complex biological systems with intricate molecular interactions and signal transduction. Comprehensive analysis of multiple molecules in tissues is essential for understanding these biological processes. In this imaging approach, co-reactants are derived from enzyme-catalyzed reactions that are triggered by substrate conversion and can be terminated by substrate elution. Therefore, after acquiring images of the enzymes, a simple wash and refresh of the reaction solution can reset the imaging system ( Figure 4 In fact, immersing the reaction chamber in PBS solution for 1 minute is sufficient to restore the initial ECL base This cycle allows multiple enzyme activity tests to be performed on a single tissue section.

[0082] The present application example selects pancreatic ductal adenocarcinoma (PDAC) tissue as a sample, and continuously maps the spatial activity of LOX, escin sulfhydryl oxidase (QSOX), nicotinamide adenine dinucleotide phosphate oxidase (NOX), and amiloride-sensitive amine oxidase (DAO) on a single tissue. Each of the four enzymatic reactions is triggered by its specific substrate. The process includes four cycles with a washing step between each cycle. ΔECL base The image should be blank and recorded at the beginning of each cycle to confirm system reset.

[0083] The four enzymes selected here are all involved in the production and remodeling of the extracellular matrix (ECM) of tumor tissue. LOX oxidizes lysine residues to aldehydes to form lysine-lysine cross-links, which are essential for the maturation of collagen in the ECM. QSOX catalyzes the oxidation of protein sulfhydryl groups to form disulfide bonds, ensuring that ECM proteins maintain the correct spatial structure. NOX transfers electrons from substrates to oxygen molecules, inducing oxidative stress in the ECM, and DAO catalyzes the deamination of amines, controlling the activity of inflammatory mediators in the ECM. Through multiplex imaging of enzyme activity, unique activity patterns of each enzyme were observed. Figure 4 As shown in B, LOX and QSOX exhibited similar patterns due to their association at the gene expression level. In contrast, active NOX had a limited distribution because it usually functions intracellularly, while DAO activity covered a large area, likely due to widespread amine metabolism disturbances in the tumor microenvironment. This result suggests that our system is capable of collecting both intensity and spatial information of multiple active enzymes in a short time. This capability is beneficial for spatial omics analysis of tissue sections.

[0084] Co-localization of expression and activity of four oxidases in a single tissue section. Abnormal enzyme states are associated with various diseases and are usually caused by deviations in enzyme expression and / or activity. To study this, the present application example spatially analyzed the expression and activity of four oxidases (LOX, QSOX, NOX, and DAO) in cancerous tissue and paracancerous tissue of PDAC patients. H&E staining was used to identify cancerous tissue and paracancerous tissue. Enzyme expression was displayed by immunofluorescence ( Figure 5 Middle A), enzyme activity from ΔECL image ( Figure 5 Colocalization analysis was performed to assess the spatial relationship between each pair of enzymes. Pearson correlation coefficient (PCC) values ​​were used to quantify the correlation, with higher values ​​indicating stronger correlation of molecular colocalization.

[0085] It is noteworthy that the reduced co-localization of QSOX / DAO activity in cancer tissues observed in the examples of this application may indicate an as-yet undiscovered interaction between these two proteins ( Figure 5 C in 5, D in 5). Their high PCC values ​​suggest that there may be a signal transduction connection. In view of the synergistic effect of QSOX and LOX, the present application example further performed the co-localization of the three enzymes. It was found that the PCC of QSOX / DAO / LOX based on activity was significantly reduced in cancer tissues ( Figure 5 (E). The high PCC values ​​provide evidence that these enzymes may have a common secretion or localization mechanism, meaning that they enter the extracellular environment through similar pathways or have a co-localization mechanism that allows the enzymes to be expressed in the same subcellular structure. One possibility for the reduced co-localization of enzyme activity may be related to the disruption of redox balance in the tumor microenvironment. The high redox environment in PDAC tissues can inhibit the function of DAO, thereby reducing DAO's metabolic consumption of products generated by QSOX and LOX during ECM protein synthesis. This process deserves further exploration by researchers in related fields, as it provides valuable information for the development of tumor drugs based on regulating enzyme activity.

[0086] Overall, the spatial information of enzyme activity obtained here provides new data for understanding human disease states that cannot be easily derived from expression or single enzyme activities obtained through traditional protein-based assays. In addition, the colocalization analysis of ΔECL images with immunofluorescence images and H&E staining images in this study demonstrates the potential of label-free ECL microscopy in multimodal imaging, which can reveal biological information from multiple perspectives, including tissue architecture, molecular expression, and functional activity.

[0087] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A Fenton-promoted luminol ECL imaging method for multiple enzyme activities, characterized in that: The following steps are involved: Step 1, tissue slice preparation, the tissue slice to be tested is warmed to room temperature, washed with PBS to remove OCT, and transferred to the ITO electrode surface; Step 2, preparing a Fe3O4 Nafion coating, applying a Fe3O4 Nafion dispersion on the tissue section on the ITO electrode, and drying in an oven to form a uniform Fe3O4 Nafion coating on the tissue section; Step 3, tissue section enzyme activity imaging, start the first cycle, in the first cycle, add L012 to the reaction chamber, apply a potential switch on the ITO electrode to capture ECL base1 Image, ECL background ECL recorded using EMCCD base images, as a baseline; Step 4, reapply the potential switch to capture ECL base2 Images were obtained from ECL using Image J. base2 Subtract ECL base1 And divide the result by ECL base1 To process the image to obtain ΔECL base Image, the image is qualified without light signal, add the target enzyme specific substrate to be detected into the reaction chamber, wait for 1 minute, and then apply the potential switch again to capture the ECL signal image ECL signal , images were processed using Image J, and ECL signal Image subtracted ECL base2 image, and divide the result by ECL base2 images to obtain enzyme-specific ECL enhancement images, and the ΔECL value corresponds to the target enzyme activity on the tissue section. The reaction solution was removed to stop the LOX catalytic reaction, and then the reaction chamber was washed with 100 μL PBS to remove the residual reaction solution; Step 5, sequential imaging of multiple enzymes on the tissue section, starting the second cycle by adding fresh L012, repeating steps 3 and 4 for the second cycle, adding multiple target enzymes in sequence, and repeating the cycle to obtain ΔECL images of multiple enzymes in the same tissue section area.

2. The Fenton-promoted luminol ECL imaging method of multiple enzyme activities according to claim 1, characterized in that: In the imaging method, an ITO electrode is used as a working electrode, a platinum wire is used as a counter electrode, an Ag / AgCl electrode is used as a reference electrode, a silicone O-ring is fixed on the conductive surface of the ITO electrode by polydimethylsiloxane and used as a reaction chamber, a potential switch is applied using a CHI 600E electrochemical analyzer, ECL imaging and bright field imaging are performed using an epifluorescence microscope equipped with an electron multiplying charge coupled device (EMCCD) camera, an MPI-A type ECL analyzer is used to collect cyclic voltammetry and ECL signals, and a JSM-7800F instrument is used to obtain scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) images.

3. The Fenton-promoted luminol ECL imaging method of multiple enzyme activities according to claim 1, characterized in that: In the step 2, the preparation method of the Fe3O4 Nafion dispersion is to dilute the Nafion dispersion with anhydrous ethanol to produce a 0.5% w / w dispersion, wash the Fe3O4 nanoparticles with PBS, and resuspend the Fe3O4 nanoparticles in anhydrous ethanol to form a 1% w / v suspension, and after ultrasonic treatment, mix the suspension with the Nafion dispersion in a volume ratio of 1:1, and ultrasonicate to obtain a Fe3O4-Nafion dispersion.

4. The Fenton-promoted luminol ECL imaging method of multiple enzyme activities according to claim 1, characterized in that: The Fe3O4 Nafion dispersion was applied to the tissue slice on the ITO electrode by evenly covering the surface of the tissue slice on the ITO electrode with a 10 μL sample of the Fe3O4 Nafion dispersion and drying it in an oven at 37° C. for 1 minute to form a uniform Fe3O4 Nafion coating on the tissue slice.

5. The Fenton-promoted luminol ECL imaging method of multiple enzyme activities according to claim 1, characterized in that: In step 3, the tissue section enzyme activity imaging is performed by examining the tissue section under the bright field of a microscope and recording the image using a common CCD camera.

6. The Fenton-promoted luminol ECL imaging method of multiple enzyme activities according to claim 1, characterized in that: In step 5, a specific substrate corresponding to the target enzyme is introduced to obtain a ΔECL image indicating the activity of the target enzyme. After completing the imaging of a single target enzyme activity, step 4 is implemented, the reaction is terminated and the reaction system is reset, and steps 3 and 4 are repeated to sequentially complete the imaging of multiple target enzyme activities on a single tissue section.

7. The Fenton-promoted luminol ECL imaging method of multiple enzyme activities according to claim 1, characterized in that: In step 5, the multiple enzymes include LOX, QSOX, NOX and DAO.

8. The Fenton-promoted luminol ECL imaging method of multiple enzyme activities according to claim 1, characterized in that: In step 5, four cycles are included, with a washing step between each cycle. ΔECL base Images should be blank and recorded at the beginning of each cycle to ensure that the reaction system is reset for each cycle.

9. The Fenton-promoted luminol ECL imaging method of multiple enzyme activities according to claim 1, characterized in that: In step 5, the substrates used in the four cycles are L-lysine, dithiothreitol, histamine and nicotinamide adenine dinucleotide phosphate, respectively.

10. Use of the detection method according to any one of the preceding claims 1 to 9 in imaging detection of enzyme activity in tissues.

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